Genotyping-by-Sequencing (GbS) the Highly Heterozygous Genome of Eucalyptus Provides Large Numbers of High Quality Genome-Wide SNPs

Genome-wide, high throughput genotyping has become indispensable to achieve the resolution and speed necessary for current high-end genetics and molecular breeding applications in forest trees. Genome complexity reduction combined to Next Generation Sequencing provides powerful direct genotyping of individuals for thousands of markers using universal reagents. In our lab setting we evaluated the practicability and performance of the GbS method developed by Elshire et al. (2011) (Institute of Genomic Diversity - Cornell) to genotype Eucalyptus. DNA of 24 ApeKI digested, barcoded and pooled samples per lane were genotyped in three lanes of a GAIIx: (1) 24 unrelated E. grandis trees; (2) 24 unrelated E. globulus trees and (3) Parents+22 offspring of a Eucalyptus reference pedigree. After filtering for quality and using the IGD GbS bioinformatics pipeline with a stringent MAF≥0.3, 10,861, 2,134 and 2,657 SNPs were genotyped with a call rate ≥95% in the three sets of samples respectively, providing very homogeneous genome coverage. Additionally 164,581 and 87,594 dominant presence/absence markers were scored in the E. grandis and E. globulus samples respectively. These results are consistent with the remarkable nucleotide diversity of Eucalyptus and suggest a higher diversity of E. grandis when compared to E. globulus, although some ascertainment bias might exist since a E. grandis reference genome was used to map the tags. This initial trial indicates that the GbS method not only provides an exceptional alternative to current SNP genotyping platforms but confirms its claimed simplicity, speed and wide applicability to highly diverse plant genomes.